Methods and compositions relating to improved lentiviral vector production systems

ABSTRACT

The present invention provides HIV-derived lentivectors which are multiply modified to create highly safe, efficient, and potent vectors for expressing transgenes for gene therapy. The lentiviral vectors comprise various combinations of an inactive central polypurine tract, a stuffer sequence, which may encode drug susceptibility genes, and a mutated hairpin in the 5′ leader sequence that substantially abolishes replication. These elements are provided in conjunction with other features of lentiviral vectors, such as a self-inactivating configuration for biosaftey and promoters such as the EF1α promoter as one example. Additional promoters are also described. The vectors can also comprise additional transcription enhancing elements such as the wood chuck hepatitis virus post-transcriptional regulatory element. These vectors therefore provide useful tools for genetic treatments for inherited and acquired disorders, gene-therapies for cancers and other disease, the creation of industrial and experimental production systems utilizing transformed cells, as well as for the study of basic cellular and genetic processes.

BACKGROUND OF THE INVENTION

[0001] The present application claims the benefit of U.S. ProvisionalApplication Serial No. 60/309,569 filed Aug. 2, 2001, the entire text ofwhich is herein incorporated by reference.

[0002] 1. Field of the Invention

[0003] The present invention relates to improved lentiviral vectors,their production and their safe use in gene delivery and expression ofdesired transgenes in target cells.

[0004] 2. Description of Related Art

[0005] Transfection of cells is an increasingly important method ofdelivering gene therapy and nucleic acid based treatment for a number ofdisorders. Transfection is the introduction of nucleic acids intorecipient eukaryotic cells and the subsequent integration of the nucleicacid sequence into chromosomal DNA. Efficient transfection requiresvectors, which facilitate the introduction of foreign nucleic acids intothe desired cells, may provide mechanisms for chromosomal integration,and provide for the appropriate expression of the traits or proteinsencoded by those nucleic acids. The design and construction ofefficient, reliable, and safe vectors for cell transfection continues tobe a substantial challenge to gene therapy and treatment methods.

[0006] Viruses of many types have formed the basis for vectors. Virusinfection involves the introduction of the viral genome into the hostcell. That property is co-opted for use as a gene delivery vehicle inviral based vectors. The viruses used are often derived from pathogenicviral species that already have many of the necessary traits andabilities to transfect cells. However, not all viruses will successfullytransfect all cell types at all stages of the cell cycle. Thus, in thedevelopment of viral vectors, viral genomes are often modified toenhance their utility and effectiveness for introducing foreign geneconstructs (transgenes) or other nucleic acids. At the same time,modifications may be introduced that reduce or eliminate their abilityto cause disease.

[0007] Lentiviruses are a subgroup of retroviruses that can infectnondividing cells owing to the karyophilic properties of theirpreintegration complex, which allow for its active import through thenucleopore. Correspondingly, lentiviral vectors derived from humanimmunodeficiency virus type 1 (HIV-1) can mediate the efficientdelivery, integration and long-term expression of transgenes intonon-mitotic cells both in vitro and in vivo (Naldini et al., 1996a;Naldini et al, 1996b; Blomer et al., 1997). For example, HIV-basedvectors can efficiently transduce human CD34⁺ hematopoietic cells in theabsence of cytokine stimulation (Akkina et al., 1996; Sutton et al.,1998; Uchida et al., 1998; Miyoshi et al., 1999; Case et al., 1999), andthese cells are capable of long-term engraftment in NOD/SCID mice(Miyoshi et al., 1999). Furthermore, bone marrow from these primaryrecipients can repopulate secondary mice with transduced cells,confirming the lentivector-mediated genetic modification of veryprimitive hematopoietic precursors, most probably bona fide stem cells.Since none of the other currently available gene delivery systems hassuch an ability, lentiviral vectors provide a previously unexploredbasis for the study of hematopoiesis and similar phenomena, and for thegene therapy of inherited and acquired disorders via the geneticmodification of human stem cells (HCLs).

[0008] This important capability is subject to significant biosafetyconcerns (Akkina et al., 1996; Sutton et al., 1998; Uchida et al.,1998). The accidental generation of replication-competent recombinants(RCRs) during the production of lentiviral vector stocks represents oneof the major problems to be solved before lentiviral vectors can beconsidered for human gene therapy.

[0009] In the retroviral genome, a single RNA molecule that alsocontains all the necessary cis-acting elements carries all the codingsequences. Biosafety of a vector production system is therefore bestachieved by distributing the sequences encoding its various componentsover as many independent units as possible, to maximize the number ofcrossovers that would be required to re-create an RCR. Lentivectorparticles are generated by co-expressing the virion packaging elementsand the vector genome in host producer cells, e.g. 293 human embryonickidney cells. In the case of HIV-1-based vectors, the core and enzymaticcomponents of the virion come from HIV-1, while the envelope protein isderived from a heterologous virus, most often VSV due to the highstability and broad tropism of its G protein. The genomic complexity ofHIV, where a whole set of genes encodes virulence factors essential forpathogenesis but dispensable for transferring the virus genetic cargo,substantially aids the development of clinically acceptable vectorsystems.

[0010] Multiply attentuated packaging systems typically now compriseonly three of the nine genes of HIV-1: gag, encoding the virion mainstructural proteins, pol, responsible for the retrovirus-specificenzymes, and rev, which encodes a post-transcriptional regulatornecessary for efficient gag and pol expression (Dull, et al., 1998).From such an extensively deleted packaging system, the parental viruscannot be reconstituted, since some 60% of its genome has beencompletely eliminated. In one version of an HIV-based packaging system,Gag/Pol, Rev, VSV G and the vector are produced from four separate DNAunits. Also, the overlap between vector and helper sequences has beenreduced to a few tens of nucleotides so that opportunities forhomologous recombination are minimized.

[0011] HIV type 1 (HIV-1) based vector particles may be generated byco-expressing the virion packaging elements and the vector genome in aso-called producer cell, e.g. 293T human enbryonic kidney cells. Thesecells may be transiently transfected with a number of plasmids.Typically from three to four plasmids are employed, but the number maybe greater depending upon the degree to which the lentiviral componentsare broken up into separate units. Generally, one plasmid encodes thecore and enzymatic components of the virion, derived from HIV-1. Thisplasmid is termed the packaging plasmid. Another plasmid encodes theenvelope protein(s), most commonly the G protein of vesicular stomatitisvirus (VSV G) because of its high stability and broad tropism. Thisplasmid may be termed the envelope expression plasmid. Yet anotherplasmid encodes the genome to be transferred to the target cell, thatis, the vector itself, and is called the transfer vector. Recombinantviruses with titers of several millions of transducing units permilliliter (TU/ml) can be generated by this technique and variantsthereof. After ultracentrifugation concentrated stocks of approximately10⁹ TU/ml can be obtained.

[0012] The vector itself is the only genetic material transferred to thetarget cells. It typically comprises the transgene cassette flanked bycis-acting elements necessary for its encapsidation, reversetranscription, nuclear import and integration. As has been previouslydone with oncoretroviral vectors, lentiviral vectors have been made thatare “self-inactivating” in that they lose the transcriptional capacityof the viral long terminal repeat (LTR) once transferred to target cells(Zufferey, et al. 1998). This modification further reduces the risk ofemergence of replication competent recombinants (RCR) and avoidsproblems linked to promoter interference.

[0013] Nevertheless, experience with retroviral vectors demonstratesthat the emergence of a replication-competent retrovirus (RCR) ispossible, although a rare event even when vectors are produced by stablepackaging cell lines and components designed to provide high safety. Thepathogenic potential of RCRs is demonstrated by the induction of cancerin monkeys injected with contaminated oncoretroviral vector stocks.Consequently, the administration of retroviral vectors to human patientsis authorized only if the presence of contaminant RCRs has been excludedby a test sensitive enough to detect a single RCR in an aliquot equal to5% of the dose actually used. Creating highly safe vectors is clearlyimportant when doses equal or superior to 10¹⁰ transducing units may benecessary to reach therapeutic efficiency.

[0014] There is therefore a significant need to develop improvedlentiviruses for use as transducing vectors capable of effectivelytransducing cells and expressing desired transgenes at high levels whilemeeting biosafety requirements. Currently available lentiviral vectorproduction systems rely on the expression of packaging and vectorelements either by transient transfection or in stable cell lines.Deletion of non-essential genes from the parental virus and splitting ofthe vector system components on separate DNA units act to help minimizethe risk of emergence of RCRs. Greatest safety is achieved with thefewest, or, ideally, with zero RCR occurrence in vector production. Thepresent invention utilizes specific changes in the packaging and vectorsystem components, their methods of production and their methods of usein order to further reduce or eliminate the occurrence of RCR.

SUMMARY OF THE INVENTION

[0015] The present invention provides for compositions and methods thatimprove the biosafety of lentiviral vector production systems in such away that, if its components undergo multiple recombination eventsreconstituting the parental virus, the resulting recombinant will stillbe defective with respect to the ability to proceed through subsequentinfection and replication. The invention further improves the biosafetyof lentiviral vector production by optionally providing for drugsensitivity for any resulting recombinants.

[0016] The present invention thus concerns, in a general and overallsense, improved vectors and methods for the production thereof that aredesigned to permit the safe transfection and transduction of animalcells, particulary human cells, and more particularly hematopoieticprogenitor cells, or stem cells (hHSC). The present inventionfacilitates appropriate expression of desired transgenes in such cellsby providing effective vectors with increased safety.

[0017] The viral vectors of the present invention, therefore, may begenerally described as recombinant vectors that include at least thelentiviral gag and pol genes, that is, those genes required for virusproduction, which permit their manufacture in reasonable quantitiesusing available producer cell lines. To meet important human safetyneeds, the more preferred vectors in accordance with the presentinvention will not include any other active lentiviral genes, such asvpr, vif, vpu, nef, tat, such as where these genes have been removed orotherwise inactivated. In fact, it is preferred that the only activelentiviral genes present in the vector will be at most theaforementioned gag and pol genes, supplemented by the rev gene as may berequired for efficient cyctoplasmic export and expression of vectorgenes.

[0018] The most preferred lentiviral genes and cis-acting sequenceelements (e.g., long terminal repeats or LTRs, the psi signal, the RRE)used in preparing lentivectors in accordance with the present inventionwill be one that is human immunodeficiency virus (HIV) derived, and moreparticularly, HIV-1 derived. Thus, the gag, pol and rev genes willpreferably be HIV genes and more preferably HIV-1 genes. However, thegag, pol and rev genes and cis-acting sequence elements from otherlentiviruses may be employed for certain applications in accordance withthe present invention, including the genes and cis-acting sequenceelements of HIV-2, simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), bovine immunodeficiency virus (BIV),Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV),caprine arthritis encephalitis virus (CAEV) and the like. Suchconstructs could be useful, for example, where one desires to modifycertain cells of non-human origin. However, the HIV based vectorbackbones (i.e., HIV cis-acting sequence elements and HIV gag, pol andrev genes) will generally be preferred in connection with most aspectsof the present invention in that HIV-based constructs are the mostefficient at transduction of human cells.

[0019] The most preferred configuration of the packaging elements is onein which the gag, pol and rev genes are present. However, the need forrev may be alleviated in some designs by using cis-acting sequencesfacilitating the cytoplasmic export of incompletely spliced RNAs in theabsence of Rev, the so-called constitutive RNA export element or CTEsuch as found in Mason-Pfizer monkey virus (Bray et al., 1994).Alternatively, specific codons may be altered in the gag and pol genesto similar effect (Kotsopoulou, et al., 2000). Also, components of thepol gene such as the integrase can be provided in a trans configuration,for instance as a VPR-integrase fusion protein (Wu, et al., 2000).

[0020] In the lentivectors of the present invention it is particularlydesirable to employ mutations in the central polypurine tract (cPPT) ofthe sequence encoding the lentiviral Gag/Pol polyprotein in thepackaging plasmid such that the introduced mutations interfere withlentiviral replication relative to wild-type genome. Such constructsprovide a biosafety feature in that the nuclear import ofreplication-competent recombinants. This feature greatly minimizes therisk that (RCRs) will emerge. The cPPT/cTS region need be inactive onlyon the packaging plasmid construct to confer this safety feature.Indeed, an active copy of the cPPT/cTS region is typically provided onthe transfer vector plasmid.

[0021] It is also desirable to employ in the present invention anadditional sequence element in the packaging plasmid encoding thelentiviral Gag/Pol polyprotein in order to increase the genome length ofany potential recombinant lentiviruses such that the effects of mutationin the central polypurine tract are maximized. This feature alsominimizes the risk of producing RCRs. The long sequence element may beintroduced into the vector genome at various positions that provide formaximizing the effects of the mutations in the central polypurine tractof the sequence encoding the lentiviral Gag/Pol polyprotein. Aparticularly preferred position is between the end of the pol or gaggenes and the beginning of the RRE sequence element.

[0022] In another preferred aspect of the invention, such long sequenceelements encode one or more genes conferring susceptibility to drugscurrently used with success to treat viral infection. One such sequenceelement may include sequence that encodes a thymidinekinase, or theIRES-tk cassette. One skilled in the art will recognize, of course, thatany such drug susceptibility gene or genes, or any like construct may beemployed to similar effect.

[0023] In a further preferred aspect of the invention, the 5′ LTR R-U5region of the vector plasmid contains a set of mutations thatadditionally prevent the replication of putative viral recombinants.Such mutations preferably include changes that either destabilize orexcessively stabilize the Poly(A) hairpin motif, which leads to reducedreplication of any RCRs.

[0024] One of skill in the art will recognize that the ultimate efficacyof these various aspects of the invention will depend upon theparticular combination of aspects employed. It is preferred that themutant sequences of the Poly(A) hairpin structures in the 5′ LTR R-U5region of the vector plasmid are to be used in conjunction with otherpreferred aspects. It is also contemplated that the invention may beembodied as various combinations of the individually describedembodiments, including a combination of all disclosed embodiments, onlytwo of the disclosed embodiments, or, employed singly in the making andusing of such lentiviral vectors in the transfection and transduction ofcells. In a most preferred embodiment of the present invention all theseaspects of the present invention will be present.

[0025] The present invention describes gene transfer vehicles thatappear particularly well suited for the transduction of cells and forthe expression of transgenes in various cell types. These compositionsand methods will facilitate the safe use of lentiviral vectors for thegenetic manipulation of cells, and should be particularly useful forboth research and therapeutic applications.

[0026] It will be understood by the skilled artisan that the inventionis not limited to any one particular cell type and that one may use thelentiviral vectors and methods of the invention for the expression oftransgenes in many cell types. Some examples of cell types contemplatedinclude terminally differentiated cells such as neurons, lung cells,muscle cells, liver cells, pancreatic cells, endothelial cells, cardiaccells, skin cells, bone marrow stromal cells, ear and eye cells.Additionally, stem cells and progenitor cells such as pancreatic ductalcells, neural precursors, and mesodermal stem cells are alsocontemplated. Most notably, however, the more preferred lentivectors ofthe present invention have highly desirable features that permit thehigh level expression of transgenes in human progenitor cells whilemeeting human biosafety requirements.

[0027] It is believed that the lentivectors of the present invention maybe employed to deliver any transgene that one desires, depending on theapplication. In the case of delivery to hematopoietic progenitor cells,one will typically select a transgene that will confer a desirablefunction on such cells, including, for example, globin genes,hematopoietic growth factors, which include erythropoietin (EPO), theinterleukins (such as Interleukin-1 (IL-1), Interleukin-2 (IL-2),Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-12 (IL-12),etc.) and the colony-stimulating factors (such as granulocytecolony-stimulating factor, granulocyte/macrophage colony-stimulatingfactor, or stem-cell colony-stimulating factor), the platelet-specificintegrin αIIbβ, multidrug resistance genes, the gp91 or gp 47 genes thatare defective in patients with chronic granulomatous disease (CGD),antiviral genes rendering cells resistant to infections with pathogenssuch as human immunodeficiency virus, genes coding for blood coagulationfactors VIII or IX which are mutated in hemophiliacs, ligands involvedin T cell-mediated immune responses such as T cell antigen receptors, Bcell antigen receptors (immunoglobulins), the interleukin receptorcommon γ chain, as well as combination of T and B cell antigen receptorsalone or in combination with single chain antibodies such as ScFv, tumornecrosis factor (TNF), IL-2, IL-12, gamma interferon, CTLA4, B7 and thelike, genes expressed in tumor cells such as Melana, MAGE genes (such asMAGE-1, MAGE-3), P198, P1A, gp100 etc.

[0028] A principal application of the present invention will be toprovide for vectors that deliver desired transgenes to hematopoieticcells for a number of possible reasons. This might include, but ofcourse not be limited to, the treatment of myelosupression andneutropenias which may be caused as a result of chemotherapy orimmunosupressive therapy or infections such as AIDS, genetic disorders,cancers and the like.

[0029] Exemplary genetic disorders of hematopoietic cells that arecontemplated include sickle cell anemia, thalassemias,hemaglobinopathies, Glanzmann thrombasthenia, lysosomal storagedisorders (such as Fabry disease, Gaucher disease, Niemann-Pick disease,and Wiskott-Aldrich syndrome), severe combined immunodeficiencysyndromes (SCID), as well as diseases resulting from the lack ofsystemic production of a secreted protein, for example, coagulationfactor VIII and/or IX. In such cases, one would desire to introducetransgenes such as globin genes, hematopoietic growth factors, whichinclude erythropoietin (EPO), the interleukins (especiallyInterleukin-1, Interleukin-2, Interleukin-3, Interleukin-6,Interleukin-12, etc.) and the colony-stimulating factors (such asgranulocyte colony-stimulating factor, granulocyte/macrophagecolony-stimulating factor, or stem-cell colony-stimulating factor), theplatelet-specific integrin αIIbβ, multidrug resistance genes, the gp91or gp 47 genes which are defective in patients with chronicgranulomatous disease (CGD), antiviral genes rendering cells resistantto infections with pathogens such as human immunodeficiency virus, genescoding for blood coagulation factors VIII or IX which are mutated inhemophiliacs, ligands involved in T cell-mediated immune responses suchas T cell antigen receptors, B cell antigen receptors (immunoglobulins),the interleukin receptor common γ chain, a combination of both T and Bcell antigen receptors alone and/or in combination with single chainantibodies (ScFv), IL2, IL12, TNF, gamma interferon, CTLA4, B7 and thelike, genes expressed in tumor cells such as Melana, MAGE genes (such asMAGE-1, MAGE-3), P198, P1A, gp100 etc.

[0030] Exemplary cancers are those of hematopoietic origin, for example,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Exemplary myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML). Lymphoid malignancies which may betreated utilizing the lentivectors of the present invention include, butare not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas contemplated as candidates for treatment utilizing thelentiviral vectors of the present invention include, but are not limitedto non-Hodgkin lymphoma and variants thereof, peripheral T-celllymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-celllymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin'sdisease.

[0031] In other embodiments, the present invention is directed to hostcells that have been transduced with one of the foregoing lentivectors.It is believed that the lentivectors of the present invention can beemployed to transduce most any cell. Exemplary cells include but are notlimited to a CD4⁺ T cell, a peripheral blood lymphocyte cell, aperipheral blood mononuclear cell, a hematopoietic stem cell, a fetalcord blood cell, a fibroblast cell, a brain cell, a lung cell, a livercell, a muscle cell, a pancreatic cell, an endothelial cell, a cardiaccell, a skin cell, a bone marrow stromal cell, and an eye cells, apancreatic ductal cell, a neural precursor, a mesodermal stem cell andthe like. The cells transduced may further be primate, murine, porcine,or human in origin, or come from another animal species.

[0032] For the production of virus particles, one may employ any cellthat is compatible with the expression of lentiviral Gag and Pol genes,or any cell that can be engineered to support such expression. Forexample, producer cells such as 293T cells, TE 671 and HT1080 cells maybe used.

[0033] Of course, as noted above, the lentivectors of the invention willbe particularly useful in the transduction of human hematopoieticprogenitor cell or a hematopoietic stem cell, obtained either from thebone marrow, the peripheral blood or the umbilical cord blood, as wellas in the tranduction of a CD4⁺ T cell, a peripheral blood B or Tlymphocyte cell, a peripheral blood mononuclear cell, a dendritic cell,and a monocytic cell. Particularly preferred targets are CD34⁺ cells.

[0034] In still other embodiments, the present invention is directed toa method for transducing a human hematopoietic stem cell comprisingcontacting a population of human cells that include hematopoietic stemcells with one of the foregoing lentivectors under conditions to effectthe transduction of a human hematopoietic progenitor cell in saidpopulation by the vector. The stem cells may be transduced in vivo or invitro, depending on the ultimate application. Even in the context ofhuman gene therapy, such as gene therapy of human stem cells, one maytransduce the stem cell in vivo or, alternatively, transduce in vitrofollowed by infusion of the transduced stem cell into a human subject.In one aspect of this embodiment, the human stem cell can be removedfrom a human, e.g., a human patient, using methods well known to thoseof skill in the art and transduced as noted above. The transduced stemcells are then reintroduced into the same or a different human.

[0035] Where a human subject is treated directly by introduction of thevector into the subject, the treatment is typically carried out byintravenous administration of the vector. When cells, for instance CD34⁺cells, dendritic cells, peripheral blood cells or tumor cells aretransduced ex vivo, the vector particles are incubated with the cellsusing a dose generally in the order of between 1 to 50 multiplicities ofinfection (MOI) which also corresponds to 1×10⁵ to 50×10⁵ transducingunits of the viral vector per 10⁵ cells. This of course includes amountof vector corresponding to 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 25,30, 35, 40, 45, and 50 MOI. Typically, the amount of vector may beexpressed in terms of HeLa transducing units (TU). Other routes forvector administration include intrarterially, endoscopically,intralesionally, percutaneously, subcutaneously, intramuscular,intrathecally, intraorbitally, intradermally, intraperitoneally,transtracheally, subcuticularly, by intrastemal injection, by inhalationor intranasal spraying, by endotracheal route and the like. Inembodiments concerning tumor/cancer therapies with the vectors of theinvention the expression vector can be delivered by direct injectioninto the tumor or into the tumor vasculature.

[0036] A typical example of ex vivo gene therapy is a patient sufferingfrom chronic granulatous disease (CGD), whose CD34⁺ cells can beisolated from the bone marrow or the peripheral blood and transduced exvivo with a lentivector expressing the gp91phox gene beforereimplantation. In the case of patients suffering from severe combinedimmunodeficiency (SCID), the inventors contemplate a similar approach,using lentivectors of the invention expressing the gene defective in thepatient, for example, the gene encoding the common gamma chain of theInterleukin receptor. For the genetic treatment of HIV infection, thepresent inventors contemplate intracellular immunization, wherein cellsare rendered resistant to the HIV virus through the introduction ofantiviral genes. In embodiments of the intracellular immunization forHIV, targets of the lentivectors of the invention include hematopoieticprogenitors, peripheral blood CD4⁺ T cells, and monocytes. As will berecognized by the skilled artisan, similar intracellular immunizationmethods can be used for other viral infections as well. For theimmunotherapy of cancers, tumor cells or antigen presenting cells suchas dendritic cells will be genetically engineered with the lentivectorsof the invention. For cancer therapies some transgenes that may be usedin the lentivector constructs of the invention are those that caninhibit, and/or kill, and/or prevent the proliferation, and/or mediatethe apoptosis of, the cancer/tumor cell and/or genes such as TNF.

[0037] The lentivectors described herein may also be used in vivo, bydirect injection into the blood or into a specific organ. For example,in one embodiment intracerebral injection of lentivectors expressing theGlial Cell Derived Nerve Growth Factor (GDNF), can be used for thetreatment of Parkinson's disease. In another example, intraportalinjection of a lentivector expressing coagulation factor VIII for thecorrection of hemophilia A is envisioned. In yet another example,intravenous or intramuscular injection of a lentivector of the presentinvention expressing the dystrophin gene for the treatment of DuchenneMuscular Dystrophy is envisioned. Thus, one of ordinary skill in the artwill appreciate the extensive use of the lentivector constructs of thepresent invention in terms of gene therapies.

[0038] As used herein the specification or claim(s) when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein “another” may mean at least asecond or more.

[0039] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0041]FIG. 1. Schematic drawing of pMDLg/pRRE and pMDLD. The cPPT/cTSsequence element (black box) is indicated on the pol gene of pMDLg/pRRE.The plasmid pMDLD is a modified version with multiple mutations in thecPTT sequence element abolishing its function. Sequence comparisonbetween the two plasmids is shown at the bottom.

[0042]FIG. 2. Inactivation of the cPPT sequence element in the packagingsystem does not affect vector production. Vectors transducing GFP wereproduced in parallel with packaging systems having or lacking afuctional cPPT sequence element by transient transfection of 293T cells.Vector stocks were matched for their reverse transcriptase activity andused to transduce 293T cells. Two days later, the percentage of GFPpositive cells was determined by FACS. Vector titers were identicalwhether the packaging system had a functional or a mutated cPPT.

[0043]FIG. 3. Strategies to increase the length of packaging plasmids.To maximize the benefit of the cPPT inactivation, genome length ofrecombinant lentiviruses must be as long as possible. To this end DNAsequence can be inserted between the pol gene and the RRE sequenceelement. The inserted DNA can work either as a stuffer only or may bechosen to fulfill additional functions. One possibility is the use of agene conferring drug sensitivity to cells infected by the recombinantlentiviruses e.g. the thymidinekinase gene (tk) from the Herpes simplexvirus (HSV). An internal ribosomal entry site (IRES) is placed upstreamof the tk gene to allow its efficient expression.

[0044]FIG. 4. Mutations known for their strong inhibitory effect onHIV-1 replication were introduced in the R-U5 region of HIV-1 basedvectors transducing the GFP gene.

[0045]FIG. 5. Mutations in the R-U5 region of lentivirus vectors do notcompromise their transduction efficiency. Vector production anddetermination of GFP positive cells were as in FIG. 2. Mutation C wasfound not to affect the transduction efficacy of the vector whereasMutation A decreases the apparent titer of the vector by a factor 10.However, the lower number of GFP positive cells with the Mut A vectorreflects the fact that the mutation prevents polyadenylation at theviral LTR but does not indicate a low transduction efficacy. This pointis demonstrated in FIG. 7.

[0046]FIG. 6. Since the presence of MutA inhibits the function of theviral polyadenylation signal, the MutA was tested in a vector carryingits own polyadenylation signal, pA, and the GFP gene. Mut A vectorscarrying their own polyadenylation signal function as wild-type vectors.

[0047]FIG. 7. Apparent titers of wild-type and vectors carrying MutA anda polyadenylation signal (pA) were identical. Vector production anddetermination of GFP positive cells were as in FIG. 2.

[0048]FIG. 8. Sequence and secondary structure of mutations A and C withthe entire panel of six disclosed by Das et al. (1997).

[0049]FIG. 9. Infectivity of wild-type HIV-1 in HeLa cells. Coloredcells indicate successful infection. Each colored cell corresponds toone infection event.

[0050]FIG. 10. Substantially reduced infectivity conferred by aninactive cPPT/cTS region in conjunction with a wild-type genome length.Viral titers were adjusted so as to equalize reverse transcriptaseacitivty to those used in FIG. 9.

[0051]FIG. 11. Infectivity conferred by an inactive cPPT/cTS region inconjunction with a genome length 1470 shorter than wild type. Viraltiters were adjusted so as to equalize reverse transcriptase acitivty tothose used in FIGS. 9 and 10.

[0052]FIG. 12. Background staining of cells in the absence of virus. Theabsence of colored cells indicates the lack of false positives in theassays that produced FIGS. 9, 10, and 11.

SEQUENCE SUMMARY

[0053] SEQ ID NO:1 corresponds to positions 5296 to 5760 of the plasmidpMDL g/p RRE, derived from the HIV-1 molecular clone NL4-3 (Accessionnumber M19921) but modified to inactivate the cPPT/cTS region. Theresulting sequence differs from the wild-type in the cPPT/cTS region,positions 5432 through 5452 as indicated in FIG. 1 and described in SEQID NO:4. SEQ ID NO:2 and SEQ ID NO:3 correspond to nucleotide positions5954 through 6558, inclusive, of previously a described vector,pHR'-CMVLacZ, (Accession number AF105229), but incorporate thenucleotide sequence changes as described by Das, et al. (1997). Thesequences contain Eco RV and Bss HII restriction enzyme sites at the 5′and 3′ ends, respectively, which are useful in introducing the sequencesinto desired constructs. SEQ ID NO:5 and SEQ ID NO:6 are the sequencesof the poly(A) hairpin structures that substantially inhibit viralreplication as identified in FIG. 8 and described in Das, et al. (1997),and which are contained within SEQ ID NO:2 and SEQ ID NO:3,respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0054] While lentiviral vectors offer a great potential for gene-therapyand especially the transduction of human hematopoietic stem cells(hHSC), vectors developed so far still suffer from concerns regardingtheir biosafety. The present invention overcomes such and otherdeficiencies in the art and describes the development of HIV-derivedvectors that have improved biosafety characteristics.

[0055] The present invention provides HIV-derived vectors which aresafe, highly efficient, and very potent for expressing transgenes inhuman and animal cells, including but not limited to hematopoieticprogenitor cells as well as in all other blood cell derivatives. Thesevectors therefore provide useful tools for genetic treatments such asinherited and acquired disorders, gene-therapies for cancers especiallythe hematological cancers, as well as for the study of hematopoiesis vialentivector-mediated modification of human HSCs.

[0056] A. Lentiviral Vectors and Gene Therapy

[0057] Lentiviruses are complex retroviruses, which, in addition to thecommon retroviral genes gag, pol, and env, contain other genes withregulatory or structural function. The higher complexity enables thevirus to modulate its life cycle, as in the course of latent infection.Some examples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu, nef and tat aredeleted making the vector biologically more safe.

[0058] Lentiviral vectors offer great advantages for gene therapy. Theyintegrate stably into chromosomes of target cells which is required forlong-term expression. Further, they do not transfer viral genestherefore avoiding the problem of generating transduced cells that canbe destroyed by cytotoxic T-cells. Furthermore, they have a relativelylarge cloning capacity, sufficient for most envisioned clinicalapplications. In addition, lentiviruses, in contrast to otherretroviruses, are capable of transducing non-dividing cells. This isvery important in the context of gene-therapy for tissues such as thehematopoietic system, the brain, liver, lungs and muscle. For example,vectors derived from HIV-1 allow efficient in vivo and ex vivo delivery,integration and stable expression of transgenes into cells such aneurons, hepatocytes, and myocytes (Blomer et al., 1997; Kafri et al.,1997; Naldini et al., 1996; Naldini et al., 1998).

[0059] The lentiviral genome and the proviral DNA have the three genesfound in retroviruses: gag, pol and env, which are flanked by two longterminal repeat (LTR) sequences. The gag gene encodes the internalstructural (matrix, capsid and nucleocapsid) proteins; the pol geneencodes the RNA-directed DNA polymerase (reverse transcriptase), aprotease and an integrase; and the env gene encodes viral envelopeglycoproteins. The 5′ and 3′ LTR's serve to promote transcription andpolyadenylation of the virion RNAs, respectively. Lentiviruses haveadditional genes including vif, vpr, tat, rev, vpu, nef and vpx.

[0060] Adjacent to the 5′ LTR are sequences necessary for reversetranscription of the genome (the tRNA primer binding site) and forefficient encapsidation of viral RNA into particles (the Psi site). Ifthe sequences necessary for encapsidation (or packaging of retroviralRNA into infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

[0061] Lentiviral vectors are known in the art, see Naldini et al.,(1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998,U.S. Pat. Nos. 6,013,516; and 5,994,136 all incorporated herein byreference. In general, these vectors are plasmid-based or virus-based,and are configured to carry the essential sequences for incorporatingforeign nucleic acid, for selection and for transfer of the nucleic acidinto a host cell.

[0062] Two components are involved in making a virus-based gene deliverysystem: first, the packaging elements, encompassing the structuralproteins as well as the enzymes necessary to generate an infectiousparticle, and second, the vector itself, i.e., the genetic material tobe transferred. Biosaftey safeguards can be introduced in the design ofboth of these components. Thus, the packaging unit of the firstgeneration HIV-based vectors comprised all HIV-1 proteins except theenvelope protein (Naldini et al., 1998, 1996a). Subsequently it wasshown that the deletion of four additional viral genes that areresponsible for virulence including, vpr, vif, vpu and nef did not alterthe utility of the vector system (Zufferey et al., 1997). It was alsoshown that Tat, the main transactivator of HIV is also dispensable forthe generation of a fully efficient vector (Dull et al., 1998). Thus,the third-generation packaging unit of the HIV-based lentiviral vectorscomprise only three genes of the parental virus: gag, pol and rev, whichhelps to eliminate the possibility of reconstitution of a wild-typevirus through recombination.

[0063] This system was further improved by removing HIV transcriptionalunits from the vector (Zufferey et al., 1998). It was demonstratedtherein that introducing a deletion in the U3 region of the 3′ LTR ofthe DNA used to produce the vector RNA generated self-inactivating (SIN)vectors. During reverse transcription this deletion is transferred tothe 5′ LTR of the proviral DNA. Enough sequence was eliminated,including the removal of a TATA box, which abolished the transcriptionalactivity of the LTR, which prevents production of full-length vector RNAin transduced cells. This however did not affect vector titers or the invitro or in vivo properties of the vector.

[0064] The present invention provides several improvements to theexisting lentivectors as described above and in other parts of thisspecification. Introducing a lentivector providing a heterologous gene,such as genes to treat hematopoietic and lympho-hematopoietic disordersin this invention, into a packaging cell yields a producer cell whichreleases infectious vector particles carrying the foreign gene ofinterest.

[0065] The env gene can be derived from any virus, includingretroviruses. The env preferably is an amphotropic envelope proteinwhich allows transduction of cells of human and other species. Examplesof retroviral-derived env genes include, but are not limited to: Moloneymurine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus(HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon apeleukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) andRous sarcoma virus (RSV). Other env genes such as Vesicular stomatitisvirus (VSV) protein G (VSV G), that of hepatitis viruses and ofinfluenza also can be used.

[0066] While VSV G protein is a desirable env gene because VSV G confersbroad host range on the recombinant virus, VSV G can be deleterious tothe host cell, e.g. the packaging cell. Thus, when a gene such as thatfor VSV G is used, it is preferred to employ an inducible promotersystem so that VSV G expression can be regulated to minimize hosttoxicity when VSV G is expression is not required. For example, thetetracycline-regulated gene expression system of Gossen & Bujard, (1992)can be employed to provide for inducible expression of VSV G whentetracycline is withdrawn from the transferred cell. Thus, the tet/VP16transactivator is present on a first vector and the VSV G codingsequence is cloned downstream from a promoter controlled by tet operatorsequences on another vector.

[0067] The vector providing the viral env nucleic acid sequence isassociated operably with regulatory sequences, e.g., a promoter orenhancer. The regulatory sequence can be any eukaryotic promoter orenhancer, including for example, EF1α, PGK, the Moloney murine leukemiavirus promoter-enhancer element, the human cytomegalovirus enhancer, thevaccinia P7.5 promoter or the like (also see examples listed in Tables 1and 2 below). In some cases, such as the Moloney murine leukemia viruspromoter-enhancer element, the promoter-enhancer elements are locatedwithin or adjacent to the LTR sequences. Preferably, the regulatorysequence is one which is not endogenous to the lentivirus from which thevector is being constructed. Thus, if the vector is being made from SIV,the SIV regulatory sequence found in the SIV LTR would be replaced by aregulatory element which does not originate from SIV.

[0068] One may further target the recombinant virus by linkage of theenvelope protein with an antibody or a particular ligand for targetingto a receptor of a particular cell-type. By inserting a sequence(including a regulatory region) of interest into the viral vector, alongwith another gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target-specific. Retroviralvectors can be made target-specific by inserting, for example, aglycolipid or a protein. Targeting often is accomplished by using anantigen-binding portion of an antibody or a recombinant antibody-typemolecule, such as a single chain antibody, to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific methods to achieve delivery of aretroviral vector to a specific target.

[0069] The heterologous or foreign nucleic acid sequence, such as apolynucleotide sequence encoding a gene such as a therapeutic gene forinherited or acquired hematopoietic disorders herein, is linked operablyto a regulatory nucleic acid sequence. Preferably, the heterologoussequence is linked to a promoter, resulting in a chimeric gene.

[0070] Marker genes may be utilized to assay for the presence of thevector, and thus, to confirm infection and integration. The presence ofa marker gene ensures the selection and growth of only those host cellswhich express the inserts. Typical selection genes encode proteins thatconfer resistance to antibiotics and other toxic substances, e.g.,histidinol, puromycin, hygromycin, neomycin, methotrexate, and cellsurface markers.

[0071] The recombinant virus of the invention is capable of transferringa nucleic acid sequence into a mammalian cell. The term, “nucleic acidsequence”, refers to any nucleic acid molecule, preferably DNA, asdiscussed in detail herein. The nucleic acid molecule may be derivedfrom a variety of sources, including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such nucleic acid sequences may comprise genomicDNA which may or may not include naturally occurring introns. Moreover,such genomic DNA may be obtained in association with promoter regions,poly A sequences or other associated sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

[0072] The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titteredby standard methods used by those of skill in the art. Thus, thepackaging constructs can be introduced into human cell lines by calciumphosphate transfection, lipofection or electroporation, generallytogether with a dominant selectable marker, such as neomycin, DHFR,Glutamine synthetase or ADA, followed by selection in the presence ofthe appropriate drug and isolation of clones. The selectable marker genecan be linked physically to the packaging genes in the construct.

[0073] Stable cell lines wherein the packaging functions are configuredto be expressed by a suitable packaging cell are known. For example, seeU.S. Pat. No. 5,686,279; and Ory et al., (1996), which describepackaging cells. The packaging cells with a lentiviral vectorincorporated in them form producer cells. Producer cells are thus cellsor cell-lines that can produce or release packaged infectious viralparticles carrying the therapeutic gene of interest. These cells canfurther be anchorage dependent which means that these cells will grow,survive, or maintain function optimally when attached to a surface suchas glass or plastic. The producer cells may also be neoplasticallytransformed cells. Some examples of anchorage dependent cell lines usedas lentiviral vector packaging cell lines when the vector is replicationcompetent are HeLa or 293 cells and PERC.6 cells.

[0074] In some applications, particularly when the virus is to be usedfor gene therapy applications, it is preferable that the vector bereplication deficient (or replication defective) to avoid uncontrolledproliferation of the virus in the individual to be treated. In suchinstances mammalian cell lines are selected which have been engineered,either by modification of the producer cell's genome to encode essentialviral functions or by the co-infection of the producer cell with ahelper virus, to express proteins complementing the effect of thesequences deleted from the viral genome. For example, for HIV-1 derivedvectors, the HIV-1 packaging cell line, PSI422, may be used as describedin Corbeau, et al. (1996). Similarly, where the viral vector to beproduced is a retrovirus, the human 293-derived retroviral packagingcell line (293GPG) capable of producing high titers of retroviralparticles may be employed as described in Ory, et al. (1996). In theproduction of minimal vector systems, the producer cell is engineered(either by modification of the viral genome or by the use of helpervirus or cosmid) to complement the functions of the parent virusenabling replication and packaging into virions in the producer cellline.

[0075] Lentiviral transfer vectors Naldini et al., (1996), have beenused to infect human cells growth-arrested in vitro and to transduceneurons after direct injection into the brain of adult rats. The vectorwas efficient at transferring marker genes in vivo into the neurons andlong term expression in the absence of detectable pathology wasachieved. Animals analyzed ten months after a single injection of thevector showed no decrease in the average level of transgene expressionand no sign of tissue pathology or immune reaction (Blomer et al.,1997).

[0076] B. The cPPT/cTS Region

[0077] The introduction of foreign nucleic acids into the nucleus of acell requires importation of the nucleic acids into the nucleus throughthe nuclear membrane. Lentiviruses utilize an active nuclear importsystem, which forms the basis of their ability to replicate efficientlyin non-dividing cells. This active import system relies upon a complexseries of events including a specific modality for reversetranscription. In particular, in HIV-1, the central polypurine tract(cPPT), located within the pol gene, initiates synthesis of a downstreamplus strand while plus strand synthesis is also initiated at the 3′polypurine tract (PPT). After strand transfer of the short DNA molecule,the upstream plus strand synthesis will initiate and proceed until thecenter of the genome is reached. At the central termination sequence(cTS) the HIV-1 reverse transcriptase is ejected, (released from itstemplate), when functioning in a strand displacement mode. (Charneau, etal., 1994) The net result is a double stranded DNA molecule with astable flap, 99 nucleotides in length at the center of the genome.

[0078] This central “flap” facilitates nuclear import. (Zennou, et al.,2000). Defects in the cPPT/cTS region that prevent the efficient reversetranscription initiating at the cPPT/cTS region prevent the formation ofthe central DNA flap. The resulting DNA molecules accumulate asnon-integrated linear viral DNA outside the nucleus. (Zennou, et al.,2000). Thus, an inactive, or substantially less active cPPT/cTS regionin a lentiviral vector packaging construct, if reconstituted into anRCR, will prevent efficient nuclear import of the RCR DNA genome duringany subsequent steps towards infection. The absence of a DNA flap in anHIV-1 virus system severly impairs viral DNA nuclear import. (Zennou, etal., 2000). Importantly, Zennou, et al. show that the addition of cPPTon the transfer vector increases levels of integration by a factor offive, whereas the inactivation of the cPPT on the viral genome itselfdecreases replication by several order of magnitude. Although unknown toZennou, et al., this difference is a function of the shorter size of thevector compared to the viral genome.

[0079] The cPPT/cTS region acts in cis with the rest of the viralgenome. The region extends over 118 nucleotides in HIV-1 and exists insimilar form in other lentiviruses. The region is found at or near thecenter of all lentiviral genomes (Zennou, et al., 2000). The cPPT/cTSsequence element overlaps with the gene encoding the integrase proteinand is present in an active form in all packaging systems described todate.

[0080] Wild-type activity of the cPPT/cTS region may be effectivelyeliminated by the mutation of the underlying nucleic acid sequence so asto disrupt the activity without effecting the function of the integraseprotein, which is also encoded by that sequence and its surroundingsequence. Packaging plasmids so altered do not reduce the vector titersthat may be achieved and so retain all the benefits of any vectorproduction system in which they are incorporated.

[0081] The elimination of wild-type activity of the cPPT/cTS region fromviral packaging systems improves their biosafety by preventing theefficient nuclear import of any RCR DNA genome during any subsequentsteps towards infection. This protective effect of an inactive cPPT/cTSregion may operate in any RCR lentiviral genome. However, the protectiveeffects can be optimized or enhanced by incorporating into the packagingplasmid a stuffer sequence, whose purpose is to enlarge the eventualgenome size of any RCR that may be produced. Larger viral genomes aremore dependent upon a fully functional cPPT/cTS region for entry intothe nucleus. Thus, a larger genome size, at least the size of awild-type lentivirus such as HIV-1, is less able to enter the nucleusthrough the mechanism mediated by the cPPT/cTS sequence region.Correspondingly, in lentiviral vectors packaging plasmids whose size hasbeen shortened through the removal or modification of non-essential orvirulence encoding genes, a stuffer sequence may be inserted to enlargethe genome size, thus utilizing more effectively the protective effectsof an inactive or mutant cPPT/cTS region.

[0082] The stuffer sequence need not be of any particular sequence otherthan one which does not rescue infectivity or in any other waycontribute to virulence of any possible RCRs that might be generated.The sequence should be of a size, however, to increase the protectiveeffects of inactive or mutant cPPT/cTS regions. For a minimal packagingplasmid such as pMDLD a stuffer sequence of about 4.4 kb in sizeeffectively recreates the native genome length of a lentivirus, and thuseffectively augments the effects of mutant cPPT/cTS regions. Optimally,the stuffer sequence will be located between the pol gene and the RRE, alocation that optimizes the likely effects of a larger genome size onthe inhibition of nuclear import by mutant cPPT/cTS regions.

[0083] C. Drug Susceptibility

[0084] The biosafety benefits provided by the replication inhibitoryeffects of larger RCR genomes in conjunction with inactive cPPT/cTSregions may be further enhanced by employing drug susceptibility genes.Drug susceptibility genes encode proteins whose presence results in anyvirus incorporating/expressing the genes being susceptible totherapeutic drugs. Thus, any unintended RCR infection may bespecifically and effectively treated.

[0085] The stuffer sequence may encode such drug susceptibility genes.One particular sequence that confers drug susceptibility is thethymidine kinase gene (Zhao-Emonet et al., 1999). The expression of adrug susceptibility gene such as thymidine kinase may be driven by apromoter. One such promoter is the IRES element. Further details of theIRES element and its use as a promoter is provided below. In the currentcontext, the IRES promoter and a thymidine kinase gene may be providedas an expression cassette, which may be inserted into the packagingplasmid as the “IRES-tk” cassette. The insertion of the IRES-tk cassetteprovides both for a genome length that aids in the effectiveness of themodifications to the cPPT/cTS region and provides a “suicide” gene thatallows therapeutic treatment of any infection with RCRs (one treat theinfected patient, not the RCR itself) that are produced and infective(Zhao-Emonet et al., 1999). The tk gene of the IRES-tk cassette isderived from the Herpes simplex virus and confers susceptibility to thedrug Ganciclovir, a substrate of TK. Thus, if an RCR is generated thatis capable of infection, the resulting infected cells may be killed withGanciclovir, thereby preventing the further spread of the RCR.Similarly, cells expressing the cytosine deaminase gene can be killedwith 5-fluorocytosine (Greco, 2001).

[0086] D. The poly(A) Hairpin

[0087] The 5′ untranslated leader sequences of lentiviral genomescontain several sequence elements crucial for viral replication. Theseinclude elements essential for transcription, mRNA splicing,dimerization, packaging, and reverse transcription. Much of the functionof the regions depends upon the secondary structure of the viral RNA(Das, et al., 1997). One such structure is a hairpin that comprises thepolyadenylation signal (AAUAAA). The structure is therefore known as thepoly(A) hairpin. The poly(A) hairpin is part of the R-U5 domain of theLTR and is present in both the 5′ and 3′ ends of the proviral genome oflentiviruses.

[0088] The role of the poly(A) hairpin in replication activity has beenconserved despite the divergence in sequence among the variouslentiviruses (Das, et al., 1997). Disruption of the poly(A) hairpinstructure through mutation of the sequence involved severely inhibitsreplication activity (Das, et al., 1997). Mutant sequences of the 5′ LTRpoly(A) hairpin region can induce such defects in replication if theyare such that they either sufficiently destabilize the hairpin or act toexcessively stabilize the hairpin. For an efficient hairpin structure,the thermodynamic stability of the sequence pairings must remain withina relatively narrow limits (Das, et al., 1997).

[0089] Das, et al., (1997), incorporated herein by reference, createdseveral different mutations within the 5′ LTR poly(A) region of HIV-1and evaluated the effects of those sequence mutations on replicationactivity. Mutant A of Das, et al. (1997) stabilized the hairpinstructure to an extent sufficient to substantially inhibit wild-typereplication activity. Mutant C of Das, et al. (1997) destabilized thehairpin structure and also substantially inhibited replication activity.The sequences of Mutant A and Mutant C are provided herein as SEQ IDNO:2 and SEQ ID NO:5 for Mutant A, and SEQ ID NO:3 and SEQ ID NO:6 forMutant C, respectively.

[0090] Particular embodiments of the present invention may includeproviding a transfer vector incorporating the replication inhibiting 5′LTR poly(A) sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQID NO:6. Preferably, these sequence elements are present in combinationwith one or more aspects of the embodiments described elsewhere in thisspecification. Thus, mutant 5′ LTR poly(A) sequences may be incorporatedinto a transfer vector that is to be used in conjunction with apackaging plasmid incorporating the cPPT/cTS region displaying reducedreplication activity. Further, the transfer vector of the presentinvention may be used in conjunction with a packaging plasmid containinga stuffer sequence to further maximize the effects of the mutatedcPPT/cTS regions so employed. As indicated elsewhere in thisspecification, such a stuffer sequence may encode drug susceptibilitygenes or expression cassettes for drug susceptibility. All these aspectsof the present invention will be present in a most preferred embodimentof the present invention.

[0091] E. SIN Design

[0092] The SIN design further increases the biosafety of lentiviralvectors. A majority of the HIV LTR is comprised of the U3 sequences. TheU3 region contains the enhancer and promoter elements that modulatebasal and induced expression of the HIV genome in infected cells and inresponse to cell activation. Several of these promoter elements areessential for viral replication. Some of the enhancer elements arehighly conserved among viral isolates and have been implicated ascritical virulence factors in viral pathogenesis. The enhancer elementsmay act to influence replication rates in the different cellular targetof the virus (Marthas et al., 1993).

[0093] As viral transcription starts at the 3′ end of the U3 region ofthe 5′ LTR, those sequences are not part of the viral mRNA and a copythereof from the 3′ LTR acts as template for the generation of bothLTR's in the integrated provirus. If the 3′ copy of the U3 region isaltered in a retroviral vector construct, the vector RNA is stillproduced from the intact 5′ LTR in producer cells, but cannot beregenerated in target cells. Transduction of such a vector results inthe inactivation of both LTR's in the progeny virus. Thus, theretrovirus is self-inactivating (SIN) and those vectors are known as SINtransfer vectors.

[0094] The SIN design is described in further detail in Zufferey et al.,1998 and U.S. Pat. No. 5,994,136 both incorporated herein by reference.As described therein, there are, however, limits to the extent of thedeletion at the 3′ LTR. First, the 5′ end of the U3 region servesanother essential function in vector transfer, being required forintegration. Thus, the terminal dinucleotide and att sequence mayrepresent the 5′ boundary of the U3 sequences which can be deleted. Inaddition, some loosely defined regions may influence the activity of thedownstream polyadenylation site in the R region. Excessive deletion ofU3 sequence from the 3′ LTR may decrease polyadenylation of vectortranscripts with adverse consequences both on the titer of the vector inproducer cells and the transgene expression in target cells. On theother hand, limited deletions may not abrogate the transcriptionalactivity of the LTR in transduced cells.

[0095] The lentiviral vectors described herein carry deletions of the U3region of the 3′ LTR spanning from nucleotide −418 to −18. This is themost extensive deletion and extends as far as to the TATA box, thereforeabrogating any transcriptional activity of the LTR in transduced cells.The titer of vector in producer cells as well as transgene expression intarget cells was unaffected in these vectors. This design thereforeprovides an enormous increase in vector safety.

[0096] SIN-type vectors with such extensive deletions of the U3 regioncannot be generated for murine leukemia virus (MLV) or spleen necrosisvirus (SNV) based retroviral vectors without compromising efficiency oftransduction.

[0097] Elimination of the −418 to −18 nucleotide sequence abolishestranscriptional activity of the LTR, thereby abolishing the productionof full length vector RNA in transduced cells. In the HIV-derivedlentivectors none of the in vitro or in vivo properties were compromisedby the SIN design. Importantly, the additional biosafety features of thepresent invention may be incorporated into SIN-type vectors andnon-SIN-type vectors with equal results.

[0098] G. Posttranscriptionally Regulating Elements (PRE)

[0099] Enhancing transgene expression may be required in certainembodiments, especially those that involve lentiviral constructs of thepresent invention with modestly active promoters.

[0100] One type of PRE is an intron positioned within the expressioncassette, which can stimulate gene expression. However, introns can bespliced out during the life cycle events of a lentivirus. Hence, ifintrons are used as PRE's they may have to be placed in an oppositeorientation to the vector genomic transcript.

[0101] Posttranscriptional regulatory elements that do not rely onsplicing events offer the advantage of not being removed during theviral life cycle. Some examples are the posttranscriptional processingelement of herpes simplex virus, the posttranscriptional regulatoryelement of the hepatitis B virus (HPRE) and the woodchuck hepatitisvirus (WPRE). Of these the WPRE is most preferred as it contains anadditional cis-acting element not found in the HPRE (Donello et al.,1998). This regulatory element is positioned within the vector so as tobe included in the RNA transcript of the transgene, but downstream ofstop codon of the transgene translational unit. As demonstrated in thepresent invention and in Zufferey et al., 1999, the WPRE element is auseful tool for stimulating and enhancing gene expression of desiredtransgenes in the context of the lentiviral vectors.

[0102] The WPRE is characterized and described in U.S. Pat. No.6,136,597, incorporated herein by reference. As described therein, theWPRE is an RNA export element that mediates efficient transport of RNAfrom the nucleus to the cytoplasm. It enhances the expression oftransgenes by insertion of a cis-acting nucleic acid sequence, such thatthe element and the transgene are contained within a single transcript.Presence of the WPRE in the sense orientation was shown to increasetransgene expression by up to 7 to 10 fold. Retroviral vectors deliversequences in the form of cDNAs instead of complete intron-containinggenes as introns are generally spliced out during the sequence of eventsleading to the formation of the retroviral particle. Introns mediate theinteraction of primary transcripts with the splicing machinery. Becausethe processing of RNAs by the splicing machinery facilitates theircytoplasmic export, due to a coupling between the splicing and transportmachineries, cDNAs are often inefficiently expressed. Thus, theinclusion of the WPRE in a vector results in enhanced expression oftransgenes.

[0103] H. Nucleic Acids

[0104] One embodiment of the present invention is to transfer nucleicacids encoding a therapeutic gene, especially a gene that providestherapy for hematopoietic and lympho-hematopoietic disorders, such asthe inherited or acquired disorders described above. In one embodimentthe nucleic acids encode a full-length, substantially full-length, orfunctional equivalent form of such a gene.

[0105] Thus, in some embodiments of the present invention, the treatmentof a hematopoietic and lympho-hematopoietic disorder involves theadministration of a lentiviral vector of the invention comprising atherapeutic nucleic acid expression construct to a cell of hematopoieticorigin. It is contemplated that the hematopoietic cells take up theconstruct and express the therapeutic polypeptide encoded by nucleicacid, thereby restoring the cells normal phenotype.

[0106] A nucleic acid may be made by any technique known to one ofordinary skill in the art. Non-limiting examples of synthetic nucleicacid, particularly a synthetic oligonucleotide, include a nucleic acidmade by in vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells (see for example,Sambrook et al. 1989, incorporated herein by reference).

[0107] A nucleic acid may be purified on polyacrylamide gels, cesiumchloride centrifugation gradients, or by any other means known to one ofordinary skill in the art (see for example, Sambrook et al. 1989,incorporated herein by reference).

[0108] The term “nucleic acid” will generally refer to at least onemolecule or strand of DNA, RNA or a derivative or mimic thereof,comprising at least one nucleobase, such as, for example, a naturallyoccurring purine or pyrimidine base found in DNA (e.g., adenine “A,”guanine “G,” thymine “T,” and cytosine “C”) or RNA (e.g. A, G, uracil“U,” and C). The term “nucleic acid” encompasses the terms“oligonucleotide” and “polynucleotide.” The term “oligonucleotide”refers to at least one molecule of between about 3 and about 100nucleobases in length. The term “polynucleotide” refers to at least onemolecule of greater than about 100 nucleobases in length. Thesedefinitions generally refer to at least one single-stranded molecule,but in specific embodiments will also encompass at least one additionalstrand that is partially, substantially or fully complementary to the atleast one single-stranded molecule. Thus, a nucleic acid may encompassat least one double-stranded molecule or at least one triple-strandedmolecule that comprises one or more complementary strand(s) or“complement(s)” of a particular sequence comprising a strand of themolecule.

[0109] In certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In other particularaspects, the gene comprises a nucleic acid, and/or encodes a polypeptideor peptide-coding sequences of a gene that is defective or mutated in ahematopoietic and lympho-hematopoietic disorder. In keeping with theterminology described herein, an “isolated gene” may comprisetranscribed nucleic acid(s), regulatory sequences, coding sequences, orthe like, isolated substantially away from other such sequences, such asother naturally occurring genes, regulatory sequences, polypeptide orpeptide encoding sequences, etc. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid comprising a nucleotidesequence that is transcribed, and the complement thereof. In particularaspects, the transcribed nucleotide sequence comprises at least onefunctional protein, polypeptide and/or peptide encoding unit. As will beunderstood by those in the art, this functional term “gene” includesboth genomic sequences, RNA or cDNA sequences, or smaller engineerednucleic acid segments, including nucleic acid segments of anon-transcribed part of a gene, including but not limited to thenon-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike. Thus, a “truncated gene” refers to a nucleic acid sequence that ismissing a stretch of contiguous nucleic acid residues.

[0110] Various nucleic acid segments may be designed based on aparticular nucleic acid sequence, and may be of any length. By assigningnumeric values to a sequence, for example, the first residue is 1, thesecond residue is 2, etc., an algorithm defining all nucleic acidsegments can be created:

n to n+y

[0111] where n is an integer from 1 to the last number of the sequenceand y is the length of the nucleic acid segment minus one, where n+ydoes not exceed the last number of the sequence. Thus, for a 10-mer, thenucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . .. and/or so on. For a 15-mer, the nucleic acid segments correspond tobases 1 to 15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, thenucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . .and/or so on.

[0112] The nucleic acid(s) of the present invention, regardless of thelength of the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The overall length may vary considerably between nucleicacid constructs. Thus, a nucleic acid segment of almost any length maybe employed, with the total length preferably being limited by the easeof preparation or use in the intended recombinant nucleic acid protocol.

[0113] The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. Vectors of thepresent invention are lentivirus based as described above and in otherparts of the specification. The nucleic acid molecules carried by thevectors of the invention encode therapeutic genes and will be used forcarrying out gene-therapies. One of skill in the art would be wellequipped to construct such a therapeutic vector through standardrecombinant techniques (see, for example, Maniatis et al., 1988 andAusubel et al., 1994, both incorporated herein by reference).

[0114] The term “expression vector” refers to any type of geneticconstruct comprising a nucleic acid coding for a RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described below.

[0115] (a) Promoters and Enhancers

[0116] A “promoter” is a control sequence that is a region of a nucleicacid sequence at which initiation and rate of transcription arecontrolled. It may contain genetic elements at which regulatory proteinsand molecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

[0117] A promoter generally comprises a sequence that functions toposition the start site for RNA synthesis. The best known example ofthis is the TATA box, but in some promoters lacking a TATA box, such as,for example, the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 late genes, a discreteelement overlying the start site itself helps to fix the place ofinitiation. Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. To bring a coding sequence “under the control of” apromoter, one positions the 5′ end of the transcription initiation siteof the transcriptional reading frame “downstream” of (i.e., 3′ of) thechosen promoter. The “upstream” promoter stimulates transcription of theDNA and promotes expression of the encoded RNA.

[0118] The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

[0119] A promoter may be one naturally associated with a nucleic acidsequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a nucleic acid sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding nucleic acid segmentunder the control of a recombinant or heterologous promoter, whichrefers to a promoter that is not normally associated with a nucleic acidsequence in its natural environment. A recombinant or heterologousenhancer refers also to an enhancer not normally associated with anucleic acid sequence in its natural environment. Such promoters orenhancers may include promoters or enhancers of other genes, andpromoters or enhancers isolated from any other virus, or prokaryotic oreukaryotic cell, and promoters or enhancers not “naturally occurring,”i.e., containing different elements of different transcriptionalregulatory regions, and/or mutations that alter expression. For example,promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. In addition to producing nucleic acid sequences ofpromoters and enhancers synthetically, sequences may be produced usingrecombinant cloning and/or nucleic acid amplification technology,including PCR™, in connection with the compositions disclosed herein(see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein byreference). Furthermore, it is contemplated the control sequences thatdirect transcription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well. Control sequences comprising promoters, enhancers andother locus or transcription controlling/modulating elements are alsoreferred to as “transcriptional cassettes”.

[0120] Naturally, it will be important to employ a promoter and/orenhancer that effectively directs the expression of the DNA segment inthe organelle, cell type, tissue, organ, or organism chosen forexpression. Those of skill in the art of molecular biology generallyknow the use of promoters, enhancers, and cell type combinations forprotein expression, (see, for example Sambrook et al., 1989,incorporated herein by reference). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous for gene therapy or forapplications such as the large-scale production of recombinant proteinsand/or peptides. The promoter may be heterologous or endogenous.

[0121] Use of a T3, T7 or SP6 cytoplasmic expression system is anotherpossible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

[0122] Tables 1 lists non-limiting examples of elements/promoters thatmay be employed, in the context of the present invention, to regulatethe expression of a RNA. Table 2 provides non-limiting examples ofinducible elements, which are regions of a nucleic acid sequence thatcan be activated in response to a specific stimulus. TABLE 1 Promoterand/or Enhancer Promoter/Enhancer References Immunoglobulin Heavy ChainBanerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985;Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al.,1984; Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin LightChain Queen et al., 1983; Picard et al., 1984 T-Cell Receptor Luria etal., 1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQβ Sullivan et al., 1987 β-Interferon Goodbourn et al., 1986; Fujita etal., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC ClassII 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman et al., 1989 β-ActinKawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase (MCK)Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Omitz et al.,1987 Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Pinkert etal., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al.,1988; Campere et al., 1989 γ-Globin Bodine et al., 1987; Perez-Stable etal., 1990 β-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-rasTriesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) α₁-AntitrypsinLatimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/orType I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang etal., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 HumanSerum Amyloid A Edbrooke et al., 1989 (SAA) Troponin I (TN I) Yutzey etal., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF)Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al.,1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herret al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al.,1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka etal., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villierset al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/orVillarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson etal., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988;Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989 PapillomaVirus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie,1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et aL, 1987;Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn etal., 1989

[0123] TABLE 2 Inducible Elements Element Inducer References MT IIPhorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger etal., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al.,1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTVGlucocorticoids Huang et al., 1981; Lee (mouse mammary et al., 1981;Majors et tumor virus) al., 1983; Chandler et al., 1983; Lee et al.,1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon Poly(rI)xTavernier et al., 1983 Poly(rc) Adenovirus 5 E2 E1A Imperiale et al.,1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a StromelysinPhorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angelet al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988Disease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-MacroglobulinIL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IInterferon Blanar et al., 1989 Gene H-2κb HSP70 E1A, SV40 Large T Tayloret al., 1989, Antigen 1990a, 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis Factor PMA Hensel et al., 1989 ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

[0124] The identity of tissue-specific promoters or elements, as well asassays to characterize their activity, is well known to those of skillin the art. Non-limiting examples of such regions include the humanLIMK2 gene (Nomoto et al., 1999), the somatostatin receptor 2 gene(Kraus et al., 1998), murine epididymal retinoic acid-binding gene(Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mousealpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene(Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997),and human platelet endothelial cell adhesion molecule-1 (Almendro etal., 1996).

[0125] The lentiviral vectors of the present invention are designed,primarily, to transfect cells with a therapeutic gene under the controlof regulated eukaryotic promoters. Although the EF1α-promoter and thePGK promoter are preferred other promoter and regulatory signal elementsas described in the Tables 1 and 2 above may also be used. Additionallyany promoter/enhancer combination (as per the Eukaryotic Promoter DataBase EPDB) could also be used to drive expression of structural genesencoding the therapeutic gene of interest that is used in context withthe lentiviral vectors of the present invention. Alternatively, atissue-specific promoter for cancer gene therapy or the targeting oftumors may be employed with the lentiviral vectors of the presentinvention for treatment of cancers, especially hematological cancers.

[0126] Typically promoters and enhancers that control the transcriptionof protein encoding genes in eukaryotic cells are composed of multiplegenetic elements. The cellular machinery is able to gather and integratethe regulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

[0127] Enhancers were originally detected as genetic elements thatincreased transcription from a promoter located at a distant position onthe same molecule of DNA. This ability to act over a large distance hadlittle precedent in classic studies of prokaryotic transcriptionalregulation. Subsequent work showed that regions of DNA with enhanceractivity are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0128] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Aside from this operational distinction, enhancers andpromoters are very similar entities.

[0129] Promoters and enhancers have the same general function ofactivating transcription in the cell. They are often overlapping andcontiguous, often seeming to have a very similar modular organization.Taken together, these considerations suggest that enhancers andpromoters are homologous entities and that the transcriptional activatorproteins bound to these sequences may interact with the cellulartranscriptional machinery in fundamentally the same way.

[0130] A signal that may prove useful is a polyadenylation signal (hGH,BGH, SV40). The use of internal ribosome binding sites (IRES) elementsare used to create multigene, or polycistronic, messages. IRES elementsare able to bypass the ribosome scanning model of 5′-methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicornavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well as an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message. In particular, the IRES element may be used to drive theexpression of drug susceptibility genes such as thymidine kinase and thelike.

[0131] In any event, it will be understood that promoters are DNAelements which when positioned functionally upstream of a gene leads tothe expression of that gene. Most transgenes that will be introducedusing the lentiviral vectors of the present invention are functionallypositioned downstream of a promoter element.

[0132] A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

[0133] (b) Multiple Cloning Sites

[0134] Vectors of the present invention can include a multiple cloningsite (MCS), which is a nucleic acid region that contains multiplerestriction enzyme sites, any of which can be used in conjunction withstandard recombinant technology to digest the vector (see, for example,Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997,incorporated herein by reference.) “Restriction enzyme digestion” refersto catalytic cleavage of a nucleic acid molecule with an enzyme thatfunctions only at specific locations in a nucleic acid molecule. Many ofthese restriction enzymes are commercially available. Use of suchenzymes is widely understood by those of skill in the art. Frequently, avector is linearized or fragmented using a restriction enzyme that cutswithin the MCS to enable exogenous sequences to be ligated to thevector. “Ligation” refers to the process of forming phosphodiester bondsbetween two nucleic acid fragments, which may or may not be contiguouswith each other. Techniques involving restriction enzymes and ligationreactions are well known to those of skill in the art of recombinanttechnology.

[0135] (c) Splicing Sites

[0136] Most transcribed eukaryotic RNA molecules will undergo RNAsplicing to remove introns from the primary transcripts. Vectorscontaining genomic eukaryotic sequences may require donor and/oracceptor splicing sites to ensure proper processing of the transcriptfor protein expression (see, for example, Chandler et al., 1997, hereinincorporated by reference.)

[0137] (d) Termination Signals

[0138] The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

[0139] In eukaryotic systems, the terminator region may also comprisespecific DNA sequences that permit site-specific cleavage of the newtranscript so as to expose a polyadenylation site. This signals aspecialized endogenous polymerase to add a stretch of about 200 Aresidues (polyA) to the 3′ end of the transcript. RNA molecules modifiedwith this polyA tail appear to be more stable and are translated moreefficiently. Thus, in other embodiments involving eukaryotes, it ispreferred that that terminator comprises a signal for the cleavage ofthe RNA, and it is more preferred that the terminator signal promotespolyadenylation of the message. The terminator and/or polyadenylationsite elements can serve to enhance message levels and to minimize readthrough from the cassette into other sequences.

[0140] Terminators contemplated for use in the invention include anyknown terminator of transcription described herein or known to one ofordinary skill in the art, including but not limited to, for example,the termination sequences of genes, such as for example the bovinegrowth hormone terminator or viral termination sequences, such as forexample the SV40 terminator. In certain embodiments, the terminationsignal may be a lack of transcribable or translatable sequence, such asdue to a sequence truncation.

[0141] (e) Polyadenylation Signals

[0142] In eukaryotic gene expression, one will typically include apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Some examples include the SV40 polyadenylationsignal or the bovine growth hormone polyadenylation signal, convenientand known to function well in various target cells. Polyadenylation mayincrease the stability of the transcript or may facilitate cytoplasmictransport.

[0143] (f) Origins of Replication

[0144] In order to propagate a vector of the invention in a host cell,it may contain one or more origins of replication sites (often termed“ori”), which is a specific nucleic acid sequence at which replicationis initiated. Alternatively an autonomously replicating sequence (ARS)can be employed if the host cell is yeast.

[0145] (g) Selectable and Screenable Markers

[0146] In certain embodiments of the invention, cells transduced withthe lentivectors of the present invention may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the transduced cell permittingeasy identification of cells containing the expression vector.Generally, a selectable marker is one that confers a property thatallows for selection. A positive selectable marker is one in which thepresence of the marker allows for its selection, while a negativeselectable marker is one in which its presence prevents its selection.An example of a positive selectable marker is a drug resistance marker.

[0147] Usually the inclusion of a drug selection marker aids in thecloning and identification of transfected cells, for example, geneticconstructs that confer resistance to neomycin, puromycin, hygromycin,DHFR, GPT, zeocin and histidinol are useful selectable markers. Inaddition to markers conferring a phenotype that allows for thediscrimination of transformants based on the implementation ofconditions, other types of markers including screenable markers such asGFP, whose basis is colorimetric analysis, are also contemplated.Alternatively, screenable enzymes such as herpes simplex virus thymidinekinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.One of skill in the art would also know how to employ immunologicmarkers, possibly in conjunction with FACS analysis. The marker used isnot believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable and screenable markers are well known toone of skill in the art.

[0148] I. Host Cells

[0149] As used herein, the terms “cell,” “cell line,” and “cell culture”may be used interchangeably. All of these terms also include theirprogeny, which is any and all subsequent generations. It is understoodthat all progeny may not be identical due to deliberate or inadvertentmutations. In the context of expressing a heterologous nucleic acidsequence, “host cell” refers to a prokaryotic or eukaryotic cell, and itincludes any transformable organisms that is capable of replicating avector and/or expressing a heterologous nucleic acid encoded by thevectors of this invention. A host cell can, and has been, used as arecipient for vectors. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny. As used herein, theterms “engineered” and “recombinant” cells or host cells are intended torefer to a cell into which an exogenous nucleic acid sequence, such as,for example, a lentivector of the invention bearing a therapeutic geneconstruct, has been introduced. Therefore, recombinant cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced nucleic acid.

[0150] In certain embodiments, it is contemplated that RNAs orproteinaceous sequences may be co-expressed with other selected RNAs orproteinaceous sequences in the same host cell. Co-expression may beachieved by co-transfecting the host cell with two or more distinctrecombinant vectors. Alternatively, a single recombinant vector may beconstructed to include multiple distinct coding regions for RNAs, whichcould then be expressed in host cells transfected with the singlevector.

[0151] Host cells may be derived from prokaryotes or eukaryotes,depending upon whether the desired result is replication of the vectoror expression of part or all of the vector-encoded nucleic acidsequences. Numerous cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org). Some examplesof host cells used in this invention include but are not limited tovirus packaging cells, virus producer cells, 293T cells, humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺cells, CD4⁺ cells, and the like.

[0152] (a) Tissues and Cells

[0153] A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition and/or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to, blood (e.g., hematopoietic cells, such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺cells, CD4⁺ cells, lymphocytes and other blood lineage cells), bonemarrow, brain, stem cells, blood vessel, liver, lung, bone, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, small intestine, spleen,stomach, testes.

[0154] (b) Organisms

[0155] In certain embodiments, the host cell or tissue may be comprisedin at least one organism. In certain embodiments, the organism may be,human, primate or murine. In other embodiments the organism may be anyeukaryote or even a prokayote (e.g., a eubacteria, an archaea), as wouldbe understood by one of ordinary skill in the art (see, for example,webpage http://phylogeny.arizona.edu/tree/phylogeny.html). Somelentivectors of the invention may employ control sequences that allowthem to be replicated and/or expressed in both prokaryotic andeukaryotic cells. One of skill in the art would further understand theconditions under which to incubate all of the above described host cellsto maintain them and to permit replication of a vector. Also understoodand known are techniques and conditions that would allow large-scaleproduction of the lentivectors of the invention, as well as productionof the nucleic acids encoded by the lentivectors and their cognatepolypeptides, proteins, or peptides some of which are therapeutic genesor proteins which will be used for gene therapies.

[0156] J. Injectable Compositions and Pharmaceutical Formulations

[0157] To achieve gene-therapy using the lentiviral vector compositionsof the present invention, one would generally contact a cell in needthereof with a lentiviral vector comprising a therapeutic gene. The cellwill further be in an organism such as a human in need of the genetherapy. The routes of administration will vary, naturally, with thelocation and nature of the disease, and include, e.g., intravenous,intrarterial, intradermal, transdermal, intramuscular, intranasal,subcutaneous, percutaneous, intratracheal, intraperitoneal,intratumoral, perfusion and lavage. The cells will also sometimes beisolated from the organisms, exposed to the lentivector ex vivo, andreimplanted afterwards.

[0158] Injection of lentiviral nucleic acid constructs of the inventionmay be delivered by syringe or any other method used for injection of asolution, as long as the expression construct can pass through theparticular gauge of needle required for injection. A novel needlelessinjection system has recently been described (U.S. Pat. No. 5,846,233)having a nozzle defining an ampule chamber for holding the solution andan energy device for pushing the solution out of the nozzle to the siteof delivery. A syringe system has also been described for use in genetherapy that permits multiple injections of predetermined quantities ofa solution precisely at any depth (U.S. Pat. No. 5,846,225).

[0159] Solutions of the nucleic acids as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0160] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intraarterial, intramuscular, subcutaneous, intratumoraland intraperitoneal administration. In this connection, sterile aqueousmedia that can be employed will be known to those of skill in the art inlight of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

[0161] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0162] The compositions disclosed herein may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like.

[0163] As used herein, “carrier” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0164] The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

[0165] The terms “contacted” and “exposed,” when applied to a cell, areused herein to describe the process by which a therapeutic lentiviralvector is delivered to a target cell.

[0166] For gene therapy to discrete, solid, accessible tumors,intratumoral injection, or injection into the tumor vasculature isspecifically contemplated. Local, regional or systemic administrationalso may be appropriate. For tumors of >4 cm, the volume to beadministered will be about 4-10 ml (preferably 10 ml), while for tumorsof <4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).Multiple injections delivered as single dose comprise about 0.1 to about0.5 ml volumes. The viral particles may advantageously be contacted byadministering multiple injections to the tumor, spaced at approximately1 cm intervals. Systemic administration is preferred for conditions suchas hematological malignancies.

[0167] Continuous administration also may be applied where appropriate.Delivery via syringe or catherization is preferred. Such continuousperfusion may take place for a period from about 1-2 hours, to about 2-6hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, toabout 1-2 wk or longer following the initiation of treatment. Generally,the dose of the therapeutic composition via continuous perfusion will beequivalent to that given by a single or multiple injections, adjustedover a period of time during which the perfusion occurs.

[0168] Treatment regimens may vary as well, and often depend on type ofdisease and location of diseased tissue, and factors such as the healthand the age of the patient. The clinician will be best suited to makesuch decisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations based on lentiviral vectors of the presentinvention.

[0169] The treatments may include various “unit doses.” A unit dose isdefined as containing a predetermined-quantity of the therapeuticcomposition comprising a lentiviral vector of the present invention. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently be described in terms of transducing units (T.U.) oflentivector, as defined by titering the vector on a cell line such asHeLa or 293. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ T.U. and higher.

[0170] K. Examples

[0171] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

[0172] Materials and Methodology Employed in Examples 1 Through 3

[0173] Cell Lines and Culture Conditions

[0174] 293T, F208 and Hela P4 cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calfserum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μs/mlstreptomycin. Cell were cultured in incubators at 37° C. in a humidified5% CO₂ atmosphere.

[0175] Plasmids Construction

[0176] All plasmid modifications were done according to standardprocedures (Sambrook et al. 1989).

[0177] Plasmid pHIV(BRU) contains the full-length proviral genome ofHIV-1 strain BRU. Plasmid pcPPT-D contains the full-length genome ofHIV-1 but the cPPT/cTS sequence element is mutated as described in SEQID NO:1 and SEQ ID NO:4.

[0178] Plasmid pHIV(BRU)ΔE was constructed by replacing the SalI-BamHIfragment with the corresponding fragment from pR9ΔE. Plasmid pcPPT-D ΔEwas constructed similarly.

[0179] Plasmid pHIV(BRU) Δ 1470 was constructed by replacing theSalI-BamHI fragment with the corresponding fragment from pCMVΔ R8.91.Plasmid pcPPT-D Δ 1470 was constructed similarly.

[0180] Vector Preparation

[0181] Stocks of vector were prepared as previously described (Zufferey,et al. 1997, Zufferey, et al. 2000, incorporated herein by reference).Three or four plasmids were transiently cotransfected into 293T cells togenerate second and third generation lentiviral vector, respectively.Vector preparation and cell transduction were done in a BL2 laboratory.Reverse transcriptase activity was measured in each vector stock usingthe method described in Klages et al. (2000), incorporated herein byreference. Differences in reverse transcriptase activity, usually lessthan 15%, were corrected by dilution of the stocks with high activity.

[0182] Virus Stocks Preparation

[0183] Stocks of virus were prepared by transfecting the differentproviral plasmids into 293T cells. For pseudotyping experiments,envelope defective proviral plasmid were cotransfected with the pMD.Gplasmid encoding the VSV G protein. Reverse transcriptase activity wasmeasured in each virus stock using the method described in Klages et al.(2000). Differences in reverse transcriptase activity, usually less than15%, were corrected by dilution of the stocks with high activity.

[0184] Vector Titration

[0185] Vectors were titrated on 293T and F208 cells. Target cell (5×10⁴cells/well) were plated in each well of a 6-well tissue culture plateand incubated 24 hour in 1 ml DMEM. Vector stocks (100 μl) or 4 serial1:10 dilutions was added to 1 ml of fresh DMEM and incubated for 2 moredays. Polybrene was omitted.

[0186] Flow Cytometry Analysis

[0187] Cells were analyzed as described (Arrighi et al., 1999), on aFACScalibur (Becton-Dickinson) with slight modifications. FL-1 was usedfor GFP, FL-2 for autofluorescence. Cells were fixed with 2%paraformaldehyde for 30 minutes, and resuspended into PBS prior toanalysis. Data were analyzed using WINMDI™ software written by J.Trotter at Scripps Institute (La Jolla, Calif.) and CellQuest software(Becton-Dickinson).

[0188] Virus Titration on HeLa P4 Cells

[0189] HeLa P4 cells express human CD4 and contain a reporter transgenemade of HIV-1 LTR fused to the E. coli LacZ gene. Upon HIV-1 infectionand genome integration, the HIV-1 Tat protein is produced. This proteintrans-activates the HIV-1 LTR promoter activity resulting in highexpression level of β-galactosidase encoded by the Lac Z gene.β-galactosidase activity is detected by an histochemical staining. HIV-1infected cells acquire a blue color. The histochemical detection ofβ-galactosidase is described in Zufferey, et al. (2000).

[0190] HeLa P4 cells (5×10⁴ cells/well) were plated in each well of a6-well tissue culture plate and cultured in 1 ml DMEM for 24 hour beforebeing infected. For infection, 1 ml of cPPT deficient vector stock wasused whereas 1 ml and dilutions corresponding to 10, 5 and 1 μl wereused for the wild-type HIV-1. The number of blue foci were counted usingan inverted light microscope in wells containing less than 100 infectionevents.

EXAMPLE 1 Modification of a Lentiviral Packaging Plasmid CentralPolypurine Tract (cPPT/cTS)

[0191] Modifications to the sequence of the cPPT/cTS region may be madeso that nuclear import is severely hindered without interfering with theactivity of the pol gene of which the cPPT/cTS region is a part (Zennou,et al., 2000). Incorporated into a packaging plasmid, such sequenceswill be represented in any RCRs that may arise during the production oruse of lentiviral vectors and so will effectively inhibit the nuclearimport of these undesired RCRs. The packaging plasmid pMDLD incorporatesmodifications to the cPPT/cTS region that effectively inhibit nuclearimport of lentiviral genomes (FIG. 1).

[0192] Plasmid pMDLD is derived from pMDLg/pRRE, which has beendescribed fully elsewhere (Dull, et al., 1998, incorporated herein byreference). Briefly, pMDLg/pRRE is a CMV-driven expression plamid thatcontains only the gag and pol coding sequences from HIV-1. Additionally,a 374-bp RRE-containing sequence from HIV-1 (HXB2) is presentimmediately downstream of the pol coding sequences. An inactive cPPT/cTSregion was substituted for that of pMDLD by replacing the wild-typeAflII-BspEI fragment (positions 5296 to 5760 of the plasmid) with thecorresponding AflII-BspEI fragment of SEQ ID NO:1. The resultingsequence differs in the cPPT/cTS region, positions 5432 through 5452 asindicated in FIG. 1 and described in SEQ ID NO:4.

[0193] The mutations which inactivate the cPPT/cTS region do not impacton the function of the integrase protein. To test whether the novelpackaging systems have conserved their ability to produce HIV-1 vectors,vector production by systems with an active or an inactive cPPT/cTSregions were compared. Vectors encoding the Green Fluorescent Protein(GFP) were generated by transient co-transfection of 293T cells withfour plasmids according to previously published protocols (Zufferey, etal. 2000). The transfer vector used in these experiments was thetransfer vector plasmid pRRLCMV GFP SIN. The envelope plasmid used waspMD.G, which encodes the vesicular stomatitis virus G protein. ThepRSVrev plasmid encoding the HIV-1 Rev protein was also used.

[0194] The resulting vector stocks were assayed for reversetranscriptase activity to eliminate any difference which could resultfrom variability in transfection efficiency. Stocks with matched reversetranscriptase activity were titrated on 293T cells and F208 cells. Fortitration, 10⁵ cells were plated in each well of 6-well plates andcultured in 1 ml of medium. 24 hours after plating, cells weretransduced with 500 microliters of vector stock or of serial dilutionsof vector stocks.

[0195] The percentage of GFP-expressing cells was determined 48 hourslater by Fluorescence Activated Cell Sorting (FACS). With both celllines, we found that vector titers were independent from thefunctionality of the cPPT/cTS sequence element in the packaging plasmids(FIG. 2). Thus, the cPPT/cTS sequence element can be inactivated inplasmids for the packaging of HIV-1 based vectors without any decreasein vector titers produced. The increase in biosafety conferred by themodification can be effectively incorporated into useful methods for theproduction of lentiviral vectors.

EXAMPLE 2 Insertion of a Stuffer Sequence into the Packaging PlasmidEnhances Biosafety

[0196] The biosafety of modifications to the sequence of the cPPT/cTSregion is enhanced when the overall length of any resultant RCR genomeis sufficiently large. Such an increase in size is obtained by insertinga stuffer sequence into the packaging plasmid pMDLD described above.

[0197] To test whether the infectivity of the HIV-1 virus in the absenceof an active cPPT/cTS sequence element depends on viral genome size, wehave generated HIV-1 proviral genomes of decreasing size by removingsequence stretches encoding the envelope protein or accessory proteins.The missing genetic information was provided in trans to complement thedefective viruses. The relative infectivity of HIV 1 viruses withdifferent genome sizes was assayed on P4 cells.

[0198] The decreasing genome size did not affect the infectivity of theviruses containing a cPPT/cTS sequence element. In contrast, theinfectivity of the mutated viruses increased when the genome size wasreduced. For each genome size, we performed pairwise comparisons ofviruses with or without an active cPPT/cTS sequence element. For virusesof wild-type length, we found that the HIV-1 virus with an inactivecPPT/cTS sequence element is 200 times less infectious than itswild-type counterpart. For viruses shorter by 1470 nucleotides, thevirus with the inactive cPPT/cTS sequence element is only 70 times lessinfectious than its wild-type counterpart.

[0199] The inhibitory effect on viral replication due to the absence ofthe cPPT/cTS function increases with the viral genome size.Consequently, the size of the packaging plasmids for the production oflentiviral vectors may be increased in order to maximize the safetyimprovement obtained by the inactivation of the cPPT/cTS sequenceelement. The size of the packaging plasmids can be increased byinserting DNA at different positions. The highest safety is obtained byinserting DNA between the end of gag/pol gene and the RRE sequenceelement because DNA inserted at this position is most likely included inthe genome of a putative recombinant virus.

EXAMPLE 3 Creation of Replication Inhibiting Mutant 5′ LTR poly(A)Hairpins

[0200] Some mutations in the 5′ R-U5 region of the Long Terminal Repeat(LTR) have profound inhibitory effects on virus replication (Das, etal., 1997). Two mutated 5′ LTR poly(A) hairpin sequences (mutA and mutC,corresponding to SEQ ID NO: 5 and SEQ ID NO: 6, respectively) wereselected from a panel of altered poly(A) hairpin sequences as disclosedby Das, et al., (1997), (see FIG. 8). These two mutants were chosenbecause they have the strongest inhibitory effect on virus replication.Previous partial characterization of these mutations suggested that themutations might affect a step of viral replication that is not requiredfor the vector function.

[0201] To test the effects of these mutant sequences, mutations A and Cin the R region of the 5′ LTR were introduced into the plasmid pHR′CMVGFP SIN (Zufferey, et al., 1998) so as to replace the wild-typesequence. Vector was produced using the wild-type or mutated transfervector plasmids in combination with pCMV ΔR8.91 as packaging plasmid andpMD.G as envelope plasmid expressing the VSV G protein. The packagingplasmid pCMV ΔR8.91 is an HIV-derived packaging construct, which encodesthe HIV-1 Gag and Pol precursors, as well as the regulatory proteins Tatand Rev (Zufferey et al., 1997). Vectors stocks were produced bytransient transfection of 293T cells according to published protocol(Zufferey, et al., 1997), matched for reverse transcriptase activity andtitrated on 293T and F208 cells as described.

[0202] Substantially identical titers for wild-type and mutC vectorswere displayed. For the mutA vector, titers were apparently reduced by afactor of 10. Since the mutation A abolishes the function of the viralpolyA addition signal, the comparison was repeated using wild-type andmutated versions of the pHR′pA-GFP-I-AC plasmid in which the GFPtransgene has it own polyA signal independent from the LTR. In thissetting the titers of all three vector stocks were identical. Thesemutations in the R region of LTR can severely impair the replication ofthe HIV-1 virus without affecting the production and the transductionefficiency of an HIV-1 derived vector.

[0203] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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[0432] Zufferey, Dull, Mandel, Bukovsky, Quiroz, Naldini, Trono,“Self-inactivating lentivirus vector for safe and efficient in vivo genedelivery,” J. Virol., 72:9873-9880, 1998.

[0433] Zufferey, Donello, Trono, Hope, “Woodchuck hepatitis virusposttranscriptional regulatory element enhances expression of transgenesdelivered by retroviral vectors,” J. Virol., 73:2886-2892, 1999.

[0434] Zufferey and Trono, Current Protocols in Neuroscience: unit 4.21:“High-titer production of lentiviral vectors,” John Wiley & Sons, NewYork, 2000.

1 6 1 360 DNA Human immunodeficiency virus type 1 1 cttaagacagcagtacaaat ggcagtattc atccacaact tcaagcgccg cggtggtatt 60 ggggggtacagtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 120 gaattacaaaaacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 180 gatccactttggaaaggacc agcaaagctc ctctggaaag gtgaaggggc agtagtaata 240 caagataatagtgacataaa agtagtgcca agaagaaaag caaagatcat tagggattat 300 ggaaaacagatggcaggtga tgattgtgtg gcaagtagac aggatgagga ttaatccgga 360 2 603 DNAHuman immunodeficiency virus type 1 2 gatatccact gacctttgga tggtgctacaagctagtacc agttgagcca gataaggtag 60 aagaggccaa taaaggagag aacaccagcttgttacaccc tgtgagcctg catgggatgg 120 atgacccgga gagagaagtg ttagagtggaggtttgacag ccgcctagca tttcatcacg 180 tggcccgaga gctgcatccg gagtacttcaagaactgctg acatcgagct tgctacaagg 240 gactttccgc tggggacttt ccagggaggcgtggcctggg cgggactggg gagtggcgag 300 ccctcagatc ctgcatataa gcagctgctttttgcctgta ctgggtctct ctggttagac 360 cagatttgag cctgggagct ctctggctaactagggaacc cactgcttaa gcctcaataa 420 agcttgcctt gaggcttaag cagtgtgtgcccgtctgttg tgtgactctg gtaactagag 480 atccctcaga cccttttagt cagtgtggaaaatctctagc agtggcgccc gaacagggac 540 ttgaaagcga aagggaaacc agaggagctctctcgacgca ggactcggct tgctgaagcg 600 cgc 603 3 605 DNA Humanimmunodeficiency virus type 1 3 gatatccact gacctttgga tggtgctacaagctagtacc agttgagcca gataaggtag 60 aagaggccaa taaaggagag aacaccagcttgttacaccc tgtgagcctg catgggatgg 120 atgacccgga gagagaagtg ttagagtggaggtttgacag ccgcctagca tttcatcacg 180 tggcccgaga gctgcatccg gagtacttcaagaactgctg acatcgagct tgctacaagg 240 gactttccgc tggggacttt ccagggaggcgtggcctggg cgggactggg gagtggcgag 300 ccctcagatc ctgcatataa gcagctgctttttgcctgta ctgggtctct ctggttagac 360 cagatttgag cctgggagct ctctggctaactagggaacc cactgcttaa gcctcaataa 420 agcttgcctt gagtgcttca acgatcgtgtgcccgtctgt tgtgtgactc tggtaactag 480 agatccctca gaccctttta gtcagtgtggaaaatctcta gcagtggcgc ccgaacaggg 540 acttgaaagc gaaagggaaa ccagaggagctctctcgacg caggactcgg cttgctgaag 600 cgcgc 605 4 21 DNA Humanimmunodeficiency virus type 1 4 aacttcaagc gccgcggtgg t 21 5 45 DNAHuman immunodeficiency virus type 1 5 cactgcttaa gcctcaataa agcttgccttgaggcttaag cagtg 45 6 48 DNA Human immunodeficiency virus type 1 6cactgcttaa gcctcaataa agcttgcctt gagtgcttca acgatcgt 48

What is claimed is:
 1. A packaging plasmid comprising a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity.
 2. The packaging plasmid of claim 1, wherein the cPPT/cTS region comprises SEQ ID NO:4.
 3. The packaging plasmid of claim 1, further comprising lentiviral gag and pol genes.
 4. The packaging plasmid of claim 3, wherein the gag and pol genes are HIV gag and pol genes.
 5. The packaging plasmid of claim 4, wherein the gag and pol genes are HIV-1 gag and pol genes.
 6. The packaging plasmid of claim 1, further comprising a stuffer sequence.
 7. The packaging plasmid of claim 6, wherein the stuffer sequence encodes a drug sensitivity gene.
 8. The packaging plasmid of claim 7, wherein the drug sensitivity gene is a thymidine kinase gene.
 9. The packaging plasmid of claim 7, wherein the drug sensitivity gene is a cytosine deaminase gene.
 10. The packaging plasmid of claim 7, wherein the stuffer sequence comprises the IRES-tk cassette.
 11. The packaging plasmid of claim 1, further comprising an RRE.
 12. The packaging plasmid of claim 1, further comprising a constitutive RNA export element.
 13. The packaging plasmid of claim 11, further comprising a stuffer sequence.
 14. The packaging plasmid of claim 13, wherein the stuffer sequence is between the pol gene and the RRE.
 15. The packaging plasmid of claim 14, wherein the stuffer sequence encodes a drug sensitivity gene.
 16. The packaging plasmid of claim 15, wherein the drug sensitivity gene is a thymidinekinase gene.
 17. The packaging plasmid of claim 15, wherein the drug sensitivity gene is a cytosine deaminase gene.
 18. The packaging plasmid of claim 14, wherein the stuffer sequence comprises the IRES-tk cassette.
 19. A lentiviral transfer vector comprising: (a) an expression cassette comprising a transgene positioned under the control of a promoter that is active to promote detectable transcription of the transgene in a cell; (b) a 3′ LTR; and (c) a 5′ LTR, wherein the 5′ LTR comprises a poly(A) hairpin sequence that substantially inhibits viral replication.
 20. The lentiviral transfer vector of claim 19, wherein the 5′LTR poly (A) sequence comprises SEQ ID NO:5.
 21. The lentiviral transfer vector of claim 19, wherein the 5′LTR poly (A) sequence comprises SEQ ID NO:6.
 22. The lentiviral transfer vector of claim 19, wherein the transfer vector is a SIN-type vector.
 23. The lentiviral transfer vector of claim 19, wherein the cell is a human cell.
 24. A method for producing a recombinant lentiviral vector comprising: (a) transfecting a cell with: (i) a packaging plasmid of claim 1; (ii) an expression plasmid, which carries an env gene not endogenous to lentiviruses; and (iii) a lentiviral transfer vector to yield a producer cell; (c) culturing the producer cell in a medium; and (d) separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium.
 25. The method of claim 24, wherein the cPPT/cTS region of the packaging plasmid comprises SEQ ID NO:4.
 26. The method of claim 24, wherein the packaging plasmid further comprises lentiviral gag and pol genes.
 27. The method of claim 26, wherein the gag and pol genes are HIV gag and pol genes.
 28. The method of claim 26, wherein the gag and pol genes are HIV-1 gag and pol genes.
 29. The method of claim 24, wherein the packaging plasmid further comprises a stuffer sequence.
 30. The method of claim 29, wherein the stuffer sequence encodes a drug sensitivity gene.
 31. The method of claim 30, wherein the drug sensitivity gene is a thymidine kinase gene.
 32. The method of claim 30, wherein the drug sensitivity gene is a cytosine deaminase gene.
 33. The method of claim 30, wherein the stuffer sequence comprises the IRES-tk cassette.
 34. The method of claim 24, wherein the packaging plasmid further comprises a constitutive RNA export element.
 35. The method of claim 24, wherein the packaging plasmid further comprises an RRE.
 36. The method of claim 35, wherein the packaging plasmid further comprises a stuffer sequence.
 37. The method of claim 36, wherein the stuffer sequence is between the pol gene and the RRE of the packaging plasmid.
 38. The method of claim 37, wherein the stuffer sequence encodes a drug sensitivity gene.
 39. The method of claim 38, wherein the drug sensitivity gene is a thymidinekinase gene.
 40. The method of claim 38, wherein the drug sensitivity gene is a cytosine deaminase gene.
 41. The method of claim 37, wherein the stuffer sequence comprises the IRES-tk cassette.
 42. The method of claim 24, wherein the lentiviral transfer vector comprises a 3′ LTR and a 5′ LTR, wherein the 5′ LTR comprises a poly(A) hairpin sequence that substantially inhibits viral replication.
 43. The method of claim 42, wherein the 5′LTR poly(A) sequence comprises SEQ ID NO:5.
 44. The method of claim 42, wherein the 5′LTR poly(A) sequence comprises SEQ ID NO:6.
 45. The method of claim 24, wherein the transfer vector is a SIN-type vector.
 46. The method of claim 42, wherein the packaging plasmid further comprises an RRE.
 47. The method of claim 42, wherein the packaging plasmid further comprises a constitutive RNA export element.
 48. The method of claim 42, wherein the packaging plasmid further comprises a stuffer sequence.
 49. The method of claim 48, wherein the stuffer sequence is between the pol gene and the RRE.
 50. The method of claim 48, wherein the stuffer sequence encodes a drug sensitivity gene.
 51. The method of claim 50, wherein the drug sensitivity gene encodes a thymidinekinase.
 52. The method of claim 50, wherein the drug sensitivity gene encodes a cytosine deaminase.
 53. The method of claim 50, wherein the stuffer sequence comprises the IRES-tk cassette.
 54. The method of claim 24, wherein the lentiviral transfer vector comprises an expression cassette comprising a transgene positioned under the control of a promoter that is active to promote detectable transcription of the transgene in a cell.
 55. The method of claim 54, wherein the cell is a human cell.
 56. A method of gene therapy comprising administering to a subject in need thereof an effective amount of a vector made in accordance with claim
 24. 57. A method for transducing an animal cell comprising contacting the cell with a vector made in accordance with claim 24 under conditions to effect the transduction of the cell by the vector.
 58. The method of claim 57, wherein the cell is a human cell.
 59. The method of claim 58, wherein the cell is a hematopoietic stem cell.
 60. The method of claim 59, wherein the cell is a human CD34+ cell.
 61. The method of claim 59, wherein the cell is treated to stimulate cell proliferation without substantial loss of stem cell pluripotency.
 62. The method of claim 57, wherein the cell is transduced in vivo.
 63. The method of claim 57, wherein the cell is transduced in vitro.
 64. The method of claim 63, wherein the transduced cell is introduced into an animal subject.
 65. The method of claim 64, wherein the subject is a human subject.
 66. A method for producing a recombinant lentiviral vector comprising: (a) transfecting a cell with: (i) a packaging plasmid; (ii) an expression plasmid, which carries an env gene not endogenous to lentiviruses; and (iii) a lentiviral transfer vector of claim 19 to yield a producer cell; (c) culturing the producer cell in a medium; and (d) separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium.
 67. The method of claim 66, wherein the packaging plasmid comprises a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity
 68. The method of claim 66, wherein the packaging plasmid further comprises lentiviral gag and pol genes.
 69. The method of claim 67, wherein the gag and pol genes are HIV gag and pol genes.
 70. The method of claim 69, whereing the gag and pol genes are HIV-1 gag and pol genes.
 71. The method of claim 66, wherein the 5′LTR poly(A) sequence of the lentiviral transfer vector comprises SEQ ID NO:5.
 72. The method of claim 66, wherein the 5′LTR poly(A) sequence of the lentiviral transfer vector comprises SEQ ID NO:6.
 73. The method of claim 66, wherein the transfer vector is a SIN-type vector.
 74. The method of claim 66, wherein the packaging plasmid further comprises an RRE.
 75. The method of claim 66, wherein the packaging plasmid further comprises a constitutive RNA export element.
 76. The method of claim 66, wherein the packaging plasmid further comprises a stuffer sequence.
 77. The method of claim 76, wherein the stuffer sequence is between the pol gene and the RRE.
 78. The method of claim 76, wherein the stuffer sequence encodes a drug sensitivity gene.
 79. The method of claim 78, wherein the drug sensitivity gene encodes a thymidinekinase.
 80. The method of claim 78, wherein the drug sensitivity gene encodes a cytosine deaminase.
 81. The method of claim 78, wherein the stuffer sequence comprises the IRES-tk cassette.
 82. The method of claim 66, wherein the packaging plasmid further comprises a constitutive RNA export element.
 83. A method of gene therapy comprising administering to a subject in need thereof an effective amount of a vector made in accordance with claim
 66. 84. A method for transducing an animal cell comprising contacting the cell with a vector made in accordance with claim 66 under conditions to effect the transduction of the cell by the vector.
 85. The method of claim 84, wherein the cell is a human cell.
 86. The method of claim 85, wherein the cell is a hematopoietic stem cell.
 87. The method of claim 86, wherein the cell is a human CD34+ cell.
 88. The method of claim 86, wherein the cell is treated to stimulate cell proliferation without substantial loss of stem cell pluripotency.
 89. The method of claim 84, wherein the cell is transduced in vivo.
 90. The method of claim 84, wherein the cell is transduced in vitro.
 91. The method of claim 90, wherein the transduced cell is introduced into an animal subject.
 92. The method of claim 91, wherein the subject is a human subject.
 93. A method for producing a recombinant lentiviral vector comprising: (a) transfecting a cell with: (i) a packaging plasmid comprising a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity; (ii) an expression plasmid, which carries an env gene not endogenous to lentiviruses; and (iii) a lentiviral transfer vector comprising an expression cassette, a 3′ LTR, and a 5′ LTR, wherein the 5′ LTR comprises a poly(A) hairpin sequence that substantially inhibits viral replication and wherein the expression cassette comprises a transgene positioned under the control of a promoter that is active to promote detectable transcription of the transgene in a cell to yield a producer cell; (c) culturing the producer cell in a medium; and (d) separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium.
 94. A method of gene therapy comprising administering to a subject in need thereof an effective amount of a vector made in accordance with claim
 93. 95. A method for transducing an animal cell comprising contacting the cell with a vector made in accordance with claim 93 under conditions to effect the transduction of the cell by the vector.
 96. The method of claim 95, wherein the cell is a human cell.
 97. The method of claim 96, wherein the cell is a hematopoietic stem cell.
 98. The method of claim 97, wherein the cell is a human CD34+ cell.
 99. The method of claim 97, wherein the cell is treated to stimulate cell proliferation without substantial loss of stem cell pluripotency.
 100. The method of claim 95, wherein the cell is transduced in vivo.
 101. The method of claim 95, wherein the cell is transduced in vitro.
 102. The method of claim 101, wherein the transduced cell is introduced into an animal subject.
 103. The method of claim 102, wherein the subject is a human subject. 